High efficiency continuous countercurrent tangential negative chromatography

ABSTRACT

A system, module and method for continuous or batch single-pass countercurrent tangential chromatography are disclosed for bind/elute and negative chromatography applications. The system includes binding, washing, elution (for bind/elute), regeneration, and equilibration single-pass modules. The resin slurry flows in a continuous single pass at steady-state through each module, while corresponding buffers flow countercurrent to the slurry facilitating efficient product and impurity extraction. The module and system include retentate pumps for better process robustness and control. A resin tank configured to be reversibly isolated from the single-pass modules facilitates a closed and disposable system. The method includes receiving unpurified product solution and resin slurry, isolating the resin tank, binding product (bind/elute) or impurities (negative) to the resin slurry, washing impurities from the resin slurry, eluting and capturing pure product from the resin slurry (bind/elute), regenerating the resin slurry following elution, and providing buffer solutions to all of the single-pass steps.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of and claims priority tonon-provisional application U.S. Ser. No. 15/305,850, filed on Oct. 21,2016, and entitled “High Efficiency Continuous Countercurrent TangentialChromatography,” which is the national stage entry of PCT applicationPCT/US15/27108, filed on Apr. 22, 2015, and entitled “High EfficiencyContinuous Countercurrent Tangential Chromatography,” which claimspriority from provisional application U.S. Ser. No. 61/983,186, filed onApr. 23, 2014, and entitled “High Efficiency Continuous CountercurrentTangential Chromatography,” the entirety of which are herebyincorporated by reference herein.

FIELD OF THE INVENTION

The present invention is generally related to chromatography. Morespecifically, this invention relates to a method, system, and apparatusof tangential chromatography using countercurrent flow to facilitateseparation of the desired product and enhance efficiency of the entirechromatography process.

BACKGROUND OF THE INVENTION

There has been a significant and sustained growth in new drug productionfeaturing monoclonal antibodies and other proteins, approximately 15-20%annually. This growth is due to expanding drug pipelines, as well asmore efficient cell lines and bioreactor growth optimizations. Theannual bio-production costs are currently estimated at $2.6 billion. Oneof the most significant investments a drug manufacturer has to make isprocess chromatography (approximately 30% or $850 million annually).

Chromatography is an integral part of drug production; its purpose inthe biotechnology industry is to purify the product proteins fromcontaminating species. The industry has started to recognize that theefficiency of the chromatography steps which are used to purify theproduct proteins are no longer keeping up with production demands. Thereare multiple reasons for this.

First, no significant improvements have been made to the columnchromatography process in the past 30 years; most of the work in theindustry has been focused on new resin development. A notable exceptionis membrane chromatography which was recently adopted by the industry.

Second, upstream technology has improved tremendously in the same timeperiod—the bioreactors are larger (up to 20,000 liters), and the titersare much higher (up to 15 g/L compared with 1-2 g/L five years ago). Asa result of longer fermentation times, there are generally moreimpurities in the bioreactor effluent solution. All of the above reasonsresult in a much heavier load for the downstream purification.

Third, column chromatography has inherent physical limitations. Columnslarger than 2 meters in diameter do not scale up. The largest columns inthe market are 2 meter diameter and 40 cm bed height. They fit 1,250 Lof resin. Assuming a binding capacity of 30 g/L of resin (common ProteinA resin capacity for monoclonal antibodies), a single cycle can bind 38kg. A 20,000 L bioreactor with an output of 10 g/L would produce a loadof 200 kg. This means that the biggest column in the market would haveto run at least 6 full cycles to process a single batch. The operationcan take up to 24 hrs and can result in a significant bottleneck for themanufacturing process.

Finally, in the present marketplace, disposability in the manufacturingprocess is gaining popularity. Disposable process steps save labor, donot require cleaning validation and are easier to run for themanufacturing personnel. Strides have been made in most downstreamprocesses to have disposable systems. These include bioreactors (up to2,000 L from Xcellerex Corp.), microfiltration (KleenPak TFF technologyfrom Pall Corp.), depth filtration (POD from Millipore Corp.), sterilefiltration (all major manufacturers), tangential flow filtration (allmajor manufacturers) and membrane chromatography (Mustang, Pall Corp.,Sartobind, and Sartorius Corp.). In the past three years, disposablepre-packed chromatography columns have been brought to market byRepligen (OPUS) and W.R. Grace (ProVance). These products may provideease of use and speed in clinical manufacturing, but are generallyconsidered to be too expensive to use in commercial and large scalemanufacturing due to the inherent limitations of the column format.

U.S. Pat. Nos. 7,947,175 and 7,988,859 to Oleg Shinkazh, entitled,“Continuous Countercurrent Tangential Chromatography” and“Countercurrent Tangential Chromatography Methods, Systems andApparatus”, respectively, disclose methods, systems and apparatus for anew technique of continuous countercurrent tangential chromatography,which address some of the challenges of the prior art. U.S. Pat. Nos.7,947,175 and 7,988,859 are incorporated herein in their entirety as iffully restated. However, a method, system, and apparatus of tangentialchromatography using countercurrent flow including improvements inpressure profile, economics, productivity, robustness and reducedcomplexity would be desirable in the art.

BRIEF DESCRIPTION OF THE INVENTION

In an exemplary embodiment, a system for continuous, single-passcountercurrent tangential chromatography having multiple single-passmodules comprises a single-pass binding step module for binding productfrom an unpurified product solution with a resin slurry, a single-passwashing step module for washing impurities from the resin slurry, asingle-pass elution step module for eluting an output of the washingstage module as purified product solution, a single-pass regenerationstep module for regenerating the resin slurry, and a resin tank forcontaining the resin slurry, the resin tank being configured to bereversibly isolated from the multiple single-pass modules followingdischarge of the resin slurry from the resin tank into the multiplesingle-pass modules. Each single-pass module includes at least oneretentate pump. The resin slurry flows in a continuous single pass atsteady-state through each of the single-pass modules, and one or more ofthe single-pass modules comprises two or more stages with permeate flowdirected countercurrent to resin slurry flow within that single-passmodule.

In another exemplary embodiment, a module comprises a first input portfor receiving an input solution, a first mixer for mixing the inputsolution with a recycled solution from a second input port to produce afirst mixed output, a stage I filter for concentrating the first mixedoutput to produce stage I retentate, wherein stage I permeate exits themodule from the stage I filter via a first output port, a second mixerfor mixing the stage I retentate from the stage I filter and an optionalbuffer solution from a third input port to produce a second mixedoutput, a stage II filter in series with the stage I filter forconcentrating the second mixed output to produce stage II retentatewhich exits the module from the stage II filter via a second outputport, wherein stage II permeate exits the module from the stage IIfilter via a third output port, and at least one retentate pump. Theinput solution from input port flows through the stage I filter and thestage II filter in a single pass, and recycled solution from the thirdoutput port flows countercurrent to the input solution into the secondinput port.

In another exemplary embodiment, a method for continuous, single-passcountercurrent tangential chromatography having multiple single-passsteps comprises receiving unpurified product solution from an upstreamprocess, receiving resin slurry from a resin tank for containing theresin slurry, the resin tank being isolated following discharge of theresin slurry from the resin tank, a single-pass binding step for bindingproduct in the unpurified product solution to the resin slurry from theresin tank, a single-pass washing step for washing impurities from theresin slurry, a single-pass elution step for eluting product from theresin slurry after the washing step, capturing purified product solutionfrom the elution step, a single-pass regeneration step for cleaning theresin slurry after the elution step, and providing buffer solutions forthe single-pass steps. The resin slurry flows in a continuous singlepass at steady-state through each of the single-pass steps, and one ormore of the single-pass steps comprises two or more stages with permeateflow directed countercurrent to resin slurry flow within thatsingle-pass stage.

In another exemplary embodiment, a system for continuous, single-passcountercurrent tangential negative chromatography having multiplesingle-pass modules comprises a single-pass binding step module forbinding impurities from an unpurified product solution with a resinslurry, a single-pass washing step module for washing out a purifiedproduct solution from the resin slurry, a single-pass regeneration stepmodule for regenerating the resin slurry, and a single-passequilibration step module for equilibrating the resin slurry. The resinslurry flows in a continuous, single-pass through each of thesingle-pass modules, and one or more of the single-pass modules comprisetwo or more stages with permeate flow directed countercurrent to resinslurry flow within that single-pass module.

In another exemplary embodiment, a method for continuous, single-passcountercurrent tangential negative chromatography having multiplesingle-pass steps, comprises receiving unpurified product solution froman upstream process, receiving resin slurry from a resin source, asingle-pass binding step for binding impurities in the unpurifiedproduct solution to the resin slurry from the resin source, asingle-pass washing step for washing out a purified product solutionfrom the resin slurry, capturing the purified product solution from thebinding step and the washing step, a single-pass regeneration step forregenerating the resin slurry, a single-pass equilibration step forequilibrating the resin slurry, and providing buffer solutions for thesingle-pass steps. The resin slurry flows in a continuous single passthrough each of the single-pass steps and one or more of the single-passsteps comprise two or more stages with permeate flow directedcountercurrent to resin slurry flow within that single-pass stage.

Other features and advantages of the present invention will be apparentfrom the following more detailed description of the preferredembodiment, taken in conjunction with the accompanying drawings whichillustrate, by way of example, the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a module for countercurrenttangential chromatography, according to an embodiment of the presentdisclosure.

FIG. 2A is a schematic representation of flow in a module containing asingle stage, according to an embodiment of the present disclosure,while FIG. 2B is a diagrammatic representation of flow in a modulecontaining two stages, according to an embodiment of the presentdisclosure.

FIG. 3 is a schematic representation of a countercurrent tangentialchromatography system operating in batch mode, according to anembodiment of the present disclosure.

FIG. 4 is a schematic representation of the countercurrent tangentialchromatography system of FIG. 3, according to an embodiment of thepresent disclosure.

FIG. 5 is a schematic representation of a countercurrent tangentialchromatography system, according to an embodiment of the presentdisclosure.

FIG. 6 is a schematic representation of a countercurrent tangentialchromatography system operating in continuous mode, according to anembodiment of the present disclosure.

FIGS. 7A, 7B, and 7C detail a process of countercurrent tangentialchromatography operating in batch mode, according to an embodiment ofthe present disclosure.

FIGS. 8A and 8B detail a process of countercurrent tangentialchromatography operating in continuous mode, according to an embodimentof the present disclosure.

FIG. 9 discloses the pressure profile results of a system lacking someof the features of the present disclosure, showing a net pressuregradient from the binding to equilibration steps.

FIG. 10 discloses the pressure profiles of a system, according to anembodiment of the present disclosure, showing very similar pressureprofiles for each operational step.

FIG. 11 is a schematic representation of a countercurrent tangentialnegative chromatography system operating in continuous mode, accordingto an embodiment of the present disclosure.

FIGS. 12A and 12B detail a process of countercurrent tangential negativechromatography operating in batch mode, according to an embodiment ofthe present disclosure.

FIGS. 13A and 13B detail a process of countercurrent tangential negativechromatography operating in continuous mode, according to an embodimentof the present disclosure.

FIG. 14 discloses the results of a mathematical model of a two-stagecountercurrent tangential chromatography, according to an embodiment ofthe present disclosure, showing a ratio of buffer to resin flow-rates(gamma) vs. percent yield for various sieving coefficients.

FIG. 15 discloses the results of a mathematical model of a three-stagecountercurrent tangential chromatography system, according to anembodiment of the present disclosure, showing a ratio of buffer to resinflow-rates (gamma) vs. percent yield for various sieving coefficients.

DETAILED DESCRIPTION OF THE INVENTION

Definitions: The following terms of art shall have the below ascribedmeanings throughout this Specification.

“Binding” step or mode indicates operation during which resin andunpurified product form a reversible complex (for positivechromatography), or during which resin and impurities form a reversiblecomplex (for negative chromatography).

“Washing” step or mode indicates operation during which resin with boundproduct is washed with a washing buffer to rid the resin of impurities(for positive chromatography), or during which resin with boundimpurities is washed with a washing buffer to wash out carryover productfrom the binding step (for negative chromatography).

“Elution” step or mode indicates operation during which the complex ofresin and the product is reversed and the purified product is collected.

“Regeneration” step or mode indicates operation during which the resinis cleaned for the purpose of reuse or for later cycles.

“Equilibration” step or mode indicates operation during which the systemis equilibrated in a neutral buffer.

“Stage” indicates an interconnected tangential flow filter and mixer.

“Single-pass module” is a module that performs one of thechromatographic operations, such as binding, washing, elution,regeneration, and equilibration in a single-pass.

Provided are exemplary systems, modules and methods. Embodiments of thepresent disclosure, in comparison to systems, modules and methods notutilizing one or more features disclosed herein, provide scalable,reliable and disposable technology that utilizes a principle ofrecycling to significantly increase process efficiency, increase thescale of operation, and decrease resin costs.

In the present invention, a chromatography column is replaced by asystem comprising modules that include one or more stages. Each stageincludes a mixer interconnected with a tangential flow filter. Thechromatography resin flows through this module in a single pass, whileoperations corresponding to the operations of a standard chromatographicprocess are performed on the resin with corresponding buffers (i.e.,binding, washing, elution, regeneration, and equilibration). The buffersfor operations that utilize more than one stage are pumped into thecorresponding modules in a countercurrent direction to the flow ofresin, and permeate solutions from later stages are recycled back intoprevious stages. This creates concentration gradients in the permeatesolutions of the tangential flow filters in the countercurrent directionto resin flow, thus saving buffer volume and increasing processefficiency. In embodiments of positive chromatography (also referred toas “bind/elute chromatography” or simply as “chromatography”), thepermeate solutions from binding, washing, equilibration and regenerationoperations are put to waste. The permeate solution from the elutionoperation is the purified product stream which is collected in aseparate product tank. In embodiments of negative chromatography, thepermeate solutions from binding and washing operations are combined andcollected as product in a separate product tank, while the permeatesolutions from regeneration and equilibration operations are put towaste (elution operations are not performed).

Referring to FIG. 1, one embodiment of a module 100 for countercurrenttangential chromatography is shown (inside the dotted line). Inputsolution enters at port 101, and the input solution and any input fromport 103 are mixed inside static mixer 102. The output from the staticmixer 102 enters a tangential flow filter 104 (also described as a“stage I filter”), from which the permeate exits the module at port 105.The retentate from tangential flow filter 104 is pumped by retentatepump 114 and is fed into static mixer 106, which may receive pure bufferat port 107. The output from static mixer 106 is fed into a tangentialflow filter 108 (also described as a “stage II filter”). The retentateis pumped out via retentate pump 112. The permeate flows out of themodule at port 109. Three-way valve 111 is utilized to direct floweither to waste 113 or to port 103. The retentate from tangential flowfilter 108 exits the module at port 110.

Referring to FIG. 2A, in one embodiment, the mixture of resin andnon-purified product solution enters at the left through port 101(through mixer 102), flows through filter 104 (with permeate exiting aswaste at port 105). The retentate is pumped out via retentate pump 112.

Referring to FIG. 2B, in one embodiment, module 100 includes two stages.The static mixer 113 is called “after-binder” and is optional. Itspurpose is to provide additional residence time in the two-stage bindingstep in order to increase product binding and yield. Note thesingle-pass nature of the flow, and the fact that flow is recycled in acountercurrent direction from port 109 to port 103 via three-way valve111. Note how in this configuration, clean buffer/non-purified productsolution enters at port 107, and recycled buffer/non-purified productsolution enters at port 103.

Referring to FIG. 3, in one embodiment, a countercurrent tangentialchromatography system 300 operating in batch mode is shown. Module 100operates in the same way as shown and described in relation to FIG. 1.Input port 101 of module 100 is connected to pumps 303, 326, andretentate pump 112. Pump 303 pumps resin from first resin tank 302. Pump326 pumps resin from second resin tank 325. Pump 305 pumps unpurifiedproduct solution from input tank 304. Port 103 of module 100 isconnected via three-way valve 111 to port 109 of module 100, as shown inFIG. 1. Waste exits the system at 113. Output from port 105 is connectedto a three-way valve 307. Three-way valve 307 is connected to producttank 309 and waste 308. Port 107 receives input into module 100 via pump314, which is connected to equilibration tank 316, washing tank 318,elution tank 320 and regeneration tank 322 via valves 315, 317, 319 and321, respectively. Output from port 110 is pumped out via retentate pump112 and three-way valve 327 to the first resin tank 302 and a secondresin tank 325.

The embodiment of the system shown in FIG. 3 is designed to treat theresin using a batch-mode operation. The resin is sequentially treated bydifferent chromatographic processes (binding, washing, elution,regeneration, and equilibration) as it cycles from the first resin tank302 to the second resin tank 325 and vice versa. For example, during thefirst stage (binding), resin passes from tank 302 to tank 325 from leftto right through module 100 via pump 303. During the next stage(washing) resin passes from tank 325 to tank 302 from left to rightthrough module 100 via pump 326. The other stages (elution,regeneration, and equilibration) alternate tanks in a similar manner.The countercurrent operation during washing, elution, regeneration, andequilibration allows greater efficiency and buffer conservation.

Referring to FIG. 4, in one embodiment, a countercurrent tangentialchromatography system 400 includes the countercurrent tangentialchromatography system 300 (as previously shown in FIG. 3) and module 100(as previously shown in FIG. 1).

Referring to FIG. 5, in another embodiment, a countercurrent tangentialchromatography system 500 is shown. This embodiment differs fromembodiments depicted in FIGS. 3 and 4, by the addition of an additional(third) stage of countercurrent separation, including mixer 530, filter532, retentate pump 534, and three-way valve 531. Waste exits at 536.Addition of the third stage may increase process efficiency and decreasebuffer utilization. It is possible to add more stages (e.g., 4 stages, 5stages, 6 stages, or more) to any module. However, mathematicalmodeling, described below, indicates that the addition of more thanthree stages may result in significantly diminishing returns in terms ofprocess efficiency and buffer utilization.

Referring to FIG. 6, in one embodiment, countercurrent tangentialchromatography system 600 operates in continuous mode. Modules 610(“binding stage”), 620 (“washing stage”), 630 (“elution stage”), 640(“regeneration stage”) and 650 (“equilibration stage”) operate in ananalogous manner to the operation of module 100 shown in FIGS. 1, 2A and2B. The thick black line on modules 620, 630, 640 and 650 represent aconnection of a third output port (as shown as 109 in FIGS. 1 and 2B)and a second input port (as shown as 103 in FIGS. 1 and 2B) viathree-way valve (as shown as 111 in FIGS. 1 and 2B). These ports andthree-way valves are not shown in FIG. 6 for clarity, but they arepresent in each of modules 620, 630, 640 and 650.

Binding stage module 610 is connected at port 605 via pump 604 tonon-purified product tank 602, via pump 606 to resin tank 608, and viathree-way valve 664 and pump 663 to binding buffer tank 662. Port 609 onmodule 610 goes to waste.

Washing stage module 620 is connected at port 613 via retentate pump 612to an output port 611 of the binding stage module 610. Port 621 goes towaste. Washing buffer enters at port 627 via pump 626 from washingbuffer tank 601.

Elution stage module 630 is connected at port 625 via retentate pump 624to output port 623 of washing stage module 620. Elution buffer enters atport 637 via pump 639 from elution buffer tank 638. Purified productexits module 630 at port 627 into product storage tank 632.

Regeneration module 640 is connected at port 635 via retentate pump 634to output port 633 of module 630. Waste exits at port 643. Regenerationbuffer enters at port 645 via pump 641 from regeneration buffer tank642.

Equilibration module 650 is connected at port 649 via retentate pump 648to output port 647 of regeneration module 640. Resin is pumped out ofport 651 via retentate pump 652 into the resin storage tank 608. Whenthe system reaches steady-state, the resin storage tank is bypassed viathree way valves 665 and 666. Waste exits from module 650 at port 661.Equilibration buffer enters at port 659 via the three-way valve 658 andthe pump 657 from the equilibration buffer tank 656.

Accordingly, unlike the system of FIG. 3, which is designed to treat theresin/product in alternating batch-mode, with resin alternating betweenthe first and the second resin tanks, the system of FIG. 6 is designedto treat the resin/product in a continuous single pass at steady-state,with resin flowing continuously from the resin tank 608, through modules610, 620, 630, 640, and 650, and returning to resin tank 608. In analternate embodiment, the resin may be circulated from module 650 backto module 610, bypassing the resin tank, when the system has reachedsteady-state. Without being bound by theory, it is believed that thecontinuous nature of the system 600 shown in FIG. 6 allows a fixedamount of resin to be used for processing an essentially unlimitedamount of unpurified product, subject only to the lifetime of the resinand process time limitations.

Referring to FIGS. 7A, 7B, and 7C process 700 of countercurrenttangential chromatography operating in batch mode is shown, according toan embodiment of the present invention. Process 700 begins at step 702.The system is flushed with binding buffer, as shown in step 704. In step706, the binding stage is started (emphasis in bold). Resin andnon-purified product is pumped into the system at appropriate flowrates, as shown in step 708. The permeate solutions are discarded fromall stages as waste during the binding stage only, as shown in step 710.The resin is collected with bound product as shown in step 712.

In step 714, the washing stage is started (emphasis in bold). The systemis flushed with washing buffer, as shown in step 716. The countercurrentpermeate is recycled and utilized during the washing stage to improveprocess efficiency and conserve buffer solution according to theprinciples of the present invention, as shown in step 718. Resin ispumped with bound product back into the first stage of the system, whereit mixes with the recycled wash buffer, as shown in step 724. The washedresin with bound product is collected in the first resin tank, whilepermeate solution is discarded as waste, as shown in step 726.

In step 728, the elution stage is started (emphasis in bold). The systemis flushed with elution buffer, as shown in step 730. The countercurrentpermeate is recycled and reused during the elution stage in order toimprove process efficiency and to conserve buffer solution, as shown instep 732. Resin bound with product is pumped back into the first stageof the system, where it mixes with the recycled elution solution, asshown in step 734.

In step 736, permeate solution from the first stage is collected asproduct solution (emphasis in bold). Resin is collected in the secondresin tank, as shown in step 738.

In step 740, the regeneration stage is started (emphasis in bold). Thesystem is flushed with regeneration solution, as shown in step 742. Thecountercurrent permeate is recycled and reused during the regenerationstage, in order to improve process efficiency and to conserve buffersolution, as shown in step 744. The resin is pumped into the firststage, where it mixes with the recycled regeneration solution, as shownin step 746. The permeate solution is discarded as waste, as shown instep 748.

In step 750, the resin is collected in the first resin tank (emphasis inbold), hence completing the cycle and allowing the reuse of resin.

Finally, the equilibration process using equilibration buffer may berepeated if more cycles are required, as shown in step 752.Alternatively, equilibration process may be performed with storagesolution if the resin requires storage, as shown in step 752. Theprocess 700 ends in step 754.

Referring to FIGS. 8A and 8B, a process 800 of countercurrent tangentialchromatography operating in continuous mode is shown, according toanother embodiment of the present invention. Process 800 begins in step802. The binding stage (Module 610 of FIG. 6) is flushed with bindingbuffer, as shown in step 804. The washing stage (Module 620 of FIG. 6)is flushed with washing buffer, as shown in step 806. The elution stage(Module 630 of FIG. 6) is flushed with elution buffer, as shown in step808. The regeneration stage (Module 640 of FIG. 6) is flushed withregeneration buffer, as shown in step 810. The equilibration stage(Module 650 of FIG. 6) is flushed with equilibration buffer, as shown instep 812. Resin is fed at the appropriate flow rates into the firststage of the system (Module 610 of FIG. 6), as shown in step 814. Allbuffer solutions are fed into the appropriate stages at appropriate flowrates, as shown in step 816. When the resin concentration reachessteady-state, it is redirected and recycled back to the entrance of thesystem via the three way valve 665 and 666, as shown in step 818.Binding buffer flow is then interchanged with the unpurified productsolution, as shown in step 820. The purified product is collected fromthe elution stage (Module 630 of FIG. 6), while all other buffersolutions are discarded to waste, as shown in step 822. The entiresystem is kept running continuously until the non-purified productsolution is completely consumed, as shown in step 824. The non-purifiedproduct solution is then switched to binding buffer, as shown in step826. The purified product solution is collected in the product tankuntil UVA280 signal is close to zero, as shown in step 828. At thatpoint the resin is switched off and the binding buffer is switched on asshown in step 830. After all the resin is recovered from the system, allbuffers are shut down, as shown in step 832. The process ends andconcludes in disassembly of the apparatus (834).

In one embodiment, the resin is injected into every stage at the sameflow rate via retentate pumps (such as, for example, 112 114, 534, 612,624, 634, 648 and 652), which are installed before every static mixer.In U.S. Pat. Nos. 7,947,175 and 7,988,859, the systems described thereinuse permeate pumps to recycle buffers. As shown in FIG. 2, the permeatepumps have been removed and the permeate flow is simply equal to bufferflow rate. The hydrodynamics are stabilized by using the steady-stateflow of the retentate pumps that serve both as pumps and as quasi checkvalves that guide the flow of buffer countercurrently to resin througheach step. Since each step is acting as a closed system, the bufferpumped into each step will be the same as the resulting flow rate of thepermeate stream out of that step.

Removing the permeate pumps and introducing retentate pumps such thatthe system is no longer pressurized by the resin slurry pump, but isinstead driven by individual retentate pumps from each step improves thepressure profiles across the entire system. Referring to FIG. 9, in asystem including permeate pumps and pressurized by the resin slurrypump, according to U.S. Pat. Nos. 7,947,175 and 7,988,859, each stageoperates at a significantly different pressure. In contrast, referringto FIG. 10, and according to an embodiment of the present inventionhaving retentate pumps, operating at steady-state, and including a resintank for containing the resin slurry, the resin tank being configured tobe reversibly isolated from the modules following discharge of the resinslurry from the resin tank into the modules, the pressure profiles foreach step are very similar, and the overall pressure of the systemdecreases by nearly 70%. Significantly more robust flow conditionsreduce or eliminate the need for slurry level control, which makes theconfiguration allowing reversible isolation (bypass) of the resin tankafter the resin is ejected from the resin tank practical. In oneembodiment, bypassing the resin tank at steady-state conditionsdecreases the overall amount of the resin and increases the overallproductivity (g purified/L of resin/hr) of the system by about 30%.Isolating the resin tank may also increase aseptic control.Additionally, eliminating control of the permeate pump flow rate mayeliminate the need for digital scales, decreasing the equipment footprint and increasing system robustness.

In one embodiment, some or all of the components of the system aredisposable.

The systems and methods disclosed herein may separate and/or purify anysuitable species or substances, including, but not limited to,biologics, enzymes, proteins, peptides, small molecules, amino acids,antibiotics, enantiomers, DNA, plasmids, RNA, siRNA, vaccines,polysaccharides, viruses, prions, virus-like particles, plasma proteins,cells, stem cells, and combinations thereof. The systems and methodsdisclosed herein may be suitable for or adapted to chiral separations,gas separations, or combinations thereof.

In one embodiment, the systems and methods disclosed herein may beapplied to or incorporated in any suitable chromatographic mode,including, but not limited to, negative/flow-through chromatography,affinity chromatography, ion exchange chromatography, hydrophobicinteraction chromatography, metal affinity chromatography, mixed modechromatography, chiral chromatography, reversed phase chromatography,his-tag chromatography, size-exclusion chromatography, or combinationsthereof.

Referring to FIG. 11, in one embodiment a countercurrent tangentialchromatography system operating in continuous mode is configured fornegative chromatography. During negative chromatography, thechromatography resin binds contaminants while not binding the product.Product is recovered in the permeate from the binding and washing steps,while the contaminants are striped and washed to waste in theregeneration and equilibrium steps. Negative chromatography is wellsuited for trace contaminant removal.

Modules 610 (“binding stage”), 620 (“washing stage”), 640 (“regenerationstage”) and 650 (“equilibration stage”) operate in an analogous mannerto the operation of module 100 shown in FIGS. 1, 2A and 2B. The thickblack line on modules 620, 640 and 650 represent a connection of a thirdoutput port (as shown as 109 in FIGS. 1 and 2B) and a second input port(as shown as 103 in FIGS. 1 and 2B) via three-way valve (as shown as 111in FIGS. 1 and 2B). These ports and three-way valves are not shown inFIG. 11 for clarity, but they are present in each of modules 620, 640and 650.

The contaminants are bound to the slurry in the binding step (610),while product is collected via binding step effluent (609). Additionalproduct is also collected via washing step (620) effluent stream (621).Binding effluent stream (609) and wash effluent stream (621) are thencombined and collected in product tank (1102). Module 640 (regenerationstep) is used to regenerate the resin and module 650 (equilibrationstep) is used to equilibrate the slurry to be recycled back to thebinding step via pump 652.

Referring to FIGS. 12A and 12B, process 1200 of countercurrenttangential negative chromatography operating in batch mode is shown,according to an embodiment of the present invention. Process 1200 beginsat step 1202. The system is flushed with binding buffer, as shown instep 1204. In step 1206, the binding stage is started (emphasis inbold). Resin and non-purified product is pumped into the system atappropriate flow rates, as shown in step 1208. The permeate solutionsare collected from all stages as product, as shown in step 1210. Theresin is collected with bound impurities as shown in step 1212.

In step 1214, the washing stage is started (emphasis in bold). Thesystem is flushed with washing buffer, as shown in step 1216. Thecountercurrent permeate is recycled and utilized during the washingstage to improve process efficiency and conserve buffer solutionaccording to the principles of the present invention, as shown in step1218. Resin is pumped with bound impurities back into the first stage ofthe system, where it mixes with the recycled wash buffer, as shown instep 1224. The washed resin with bound impurities is collected in thefirst resin tank, while permeate solution is collected as product, asshown in step 1226.

In step 1240, the regeneration stage is started (emphasis in bold). Thesystem is flushed with regeneration solution, as shown in step 1242. Thecountercurrent permeate is recycled and reused during the regenerationstage, in order to improve process efficiency and to conserve buffersolution, as shown in step 1244. The resin is pumped into the firststage, where it mixes with the recycled regeneration solution, as shownin step 1246. The permeate solution is discarded as waste, as shown instep 1248.

In step 1250, the resin is collected in the first resin tank (emphasisin bold), hence completing the cycle and allowing the reuse of resin.

Finally, the equilibration process using equilibration buffer may berepeated if more cycles are required, as shown in step 1252.Alternatively, equilibration process may be performed with storagesolution if the resin requires storage, as shown in step 1252. Theprocess 1200 ends in step 1254.

Referring to FIGS. 13A and 13B, a process 1300 of countercurrenttangential negative chromatography operating in continuous mode isshown, according to another embodiment of the present invention. Process1300 begins in step 1302. The binding stage (Module 610 of FIG. 11) isflushed with binding buffer, as shown in step 1304. The washing stage(Module 620 of FIG. 11) is flushed with washing buffer, as shown in step1306. The regeneration stage (Module 640 of FIG. 11) is flushed withregeneration buffer, as shown in step 1310. The equilibration stage(Module 650 of FIG. 11) is flushed with equilibration buffer, as shownin step 1312. Resin is fed at the appropriate flow rates into the firststage of the system (Module 610 of FIG. 11), as shown in step 1314. Allbuffer solutions are fed into the appropriate stages at appropriate flowrates, as shown in step 1316. When the resin concentration reachessteady-state, it is redirected and recycled back to the entrance of thesystem via the three way valve 665 and 666, as shown in step 1318.Binding buffer flow is then interchanged with the unpurified productsolution, as shown in step 1320. The purified product is collected fromthe binding and wash stages (Modules 610 and 620 of FIG. 11), while allother buffer solutions are discarded to waste, as shown in step 1322.The entire system is kept running continuously until the non-purifiedproduct solution is completely consumed, as shown in step 1324. Thenon-purified product solution is then switched to binding buffer, asshown in step 1326. The purified product solution is collected in theproduct tank until UV 280 signal is close to zero, as shown in step1328. At the point the resin is switched off and the binding buffer isswitched on as shown in step 1330. After all the resin is recovered fromthe system, all buffers are shut down, as shown in step 1332. Theprocess ends and concludes in disassembly of the apparatus (1334).

Modeling

Product recovery is one of the most important cost drivers inchromatography. This is because the protein molecules are of extremelyhigh value. A capture chromatography process should have a recovery ofat least 90%. Therefore, it was decided to model the product recoverystage of the present invention (the elution stage).

The following assumptions were made in this model:

1. The tangential flow (TFF) membranes in the module are able to processthe slurry of resin and elution buffer at appropriate conversion factors(upwards of 80%).

2. The kinetics of desorption of the product molecule from the resin arefast.

3. The sieving coefficient of the TFF membrane for the product isconstant throughout the process.

4. The system is “dead-space” free.

The impacts on the percent yield (% recovery) of the following variablesare explored in this model:

1. “Gamma (γ)” is the ratio of elution buffer flow-rate to resin bufferflow rate, and governs the dilution of the product, buffer usage, andwashing efficiency. This variable can be controlled by the operator.

2. “s” is the sieving coefficient of the TFF membrane for the productmolecule. s equals the product concentration in the permeate divided bythe product concentration in the fluid phase in the retentate (i.e., theconcentration of unbound product in the retentate). This is an inherentproperty of the membrane and cannot be changed by the operator.

3. “N” is the number of stages; the present model explores a two-stageand a three-stage system in operation. As the number of stagesincreases, with all other variables held constant, the washingefficiency and product recovery increase. However, more stages increasethe complexity and cost of the system.

Model equations were derived by using material balances and solving for% yield. It became convenient to introduce a new variable α=γ·s.

Equation 1 shows the percent-yield for a two-stage system as a functionof α:

$\begin{matrix}{{\$ \mspace{14mu} {Yield}} = {\left( {1 - \frac{1}{\left( {1 + \alpha + \alpha^{2}} \right)}} \right) \star {100\%}}} & (1)\end{matrix}$

Equation 2 shows the percent-yield for a three-stage system as afunction of α:

$\begin{matrix}{{\% \mspace{14mu} {Yield}} = {\left( {1 - \frac{1}{\left( {1 + \alpha + \alpha^{2} + \alpha^{3}} \right)}} \right) \star {100\%}}} & (2)\end{matrix}$

FIGS. 14 and 15 show the results of this model; gamma (γ) is theindependent variable, and percent (%) yield is the dependent variable.Percent yield curves are generated for specific sieving coefficients forboth models (s=0.5, 0.7, 0.8, 1.0).

FIG. 14 shows the results for a two-stage countercurrent tangentialchromatography system showing the percent yield as a function of theratio of buffer to resin flow-rates (gamma) for sieving coefficientss=0.5, 0.7, 0.8, 1.0.

FIG. 15 shows the results for a three-stage countercurrent tangentialchromatography system showing the percent yield as a function of theratio of buffer to resin flow-rates (gamma) for sieving coefficientss=0.5, 0.7, 0.8, 1.0.

The results of the model show that greater than 95% yield can beachieved by both the two-stage and the three-stage systems. Sievingcoefficients for these processes are expected to be within a range of[0.8-1.0] because the membranes used in this system would be microporousand would therefore be expected to pass the product molecule relativelyfreely. The two-stage system would need a higher buffer to feed ratio(γ) than the three-stage system to achieve the same percent (%) yield.Therefore, the recommended operating gamma (γ) for a two-stage system is4 to 6, and for a three-stage system the recommended operating gamma (γ)is 3 to 4.

A modeling example is described herein of protein A capture of 20,000 Lbioreactor harvest, 5 g/L IgG concentration, in a three-stagecountercurrent tangential chromatography system operating in batch mode,as shown in FIG. 5. This example is illustrative of one of many modes ofoperation of the present invention.

This modeling example makes the following assumptions:

1. Residence time=0.5 min (hypothetical “small” protein A bead)

2. Resin capacity=30 g/L

3. General Electric® hollow fibers are used as the TFF membrane. Theareas and hold up volumes are used from existing large scale GeneralElectric® modules.

4. Flux=100 LMH

5. An 80% conversion factor is assumed in the TFF filters.

TABLE 1 Modeling results Volume 20000 L Binding stage time 0.175 hrsProduct conc. 5 g/L Total product 100 kg Wash Volume 4 Resin Volumes(RV) 1200 L Total Membrane area 300 m2 Washin stage Time 0.120 hrs # ofstages 3 Washing dilution factor 4 Elution Volume 4 Resin Volumes (RV)1200 L Resin Volume 300 L Elution stage Time 0.12 hrs Resin Capacity 30g/L Regeneration Buffer volume (4 RV) optional 1200 L Flux 100 LMH WashTime 0.120 hrs One cycle processes 9 kg MAB One cycle Volume 1800 LEquilibrium Buffer volume (4 RV) 1200 Wash Time 0.120 hrs Residence time0.5 min Static mixer volume 100 L Total Flow 200 L/min Resin Flow rate28.6 L/min Total Cycle time 0.66 hrs Feed Flow rate 171.4 L/min # ofcycles 12 Feed Flux 34.3 LMH Total Processing Time 7.9 hrs

The results of this model show the following:

1. 20,000 L of unpurified product can be processed with 300 L of resinwhich represents a factor of 4 decrease from conventional columnchromatography.

2. The operation can be performed in a single 8-hr shift.

3. Number of cycles can be decreased by increasing resin volume.

4. Efficiency and process time could be increased by increasing flux.

The inventor recognizes numerous and substantial advantages of thepresent invention to the downstream purification process, including:

1. Current technology could be readily adapted to this process becauseexisting components are readily available in the market. Namely, thetangential flow filters (cassettes, hollow fibers and ceramic membranes)and chromatography resins are readily available. It might beadvantageous to develop a new line of resins specifically designed forthis invention by using smaller beads than in conventional columnchromatography. This would minimize mass transfer limitations, increasedynamic binding capacity, and make the process more efficient.

2. Tangential chromatography systems according to the principles of thisinvention may be scaled as large as necessary, similarly to anytangential flow system. This is not the case with conventional columnchromatography—the largest scalable columns in the market are currentlylimited to 2 meters in diameter.

3. Continuous-mode countercurrent tangential chromatography can bedesigned as shown in FIG. 6. In general, continuous processes are moreefficient and require a smaller system size.

4. There is potential to run this system in a completely disposablemanner. This is because much smaller amounts of resin are needed forthis operation than in column chromatography (this would be true forcheaper resin kinds such as ion exchange resins). Additionally, thetangential flow filters at smaller scales could be used on a disposablebasis as well.

5. The use of resin could be an order of magnitude lower than inconventional chromatography, causing significant cost savings by as muchas 80%.

Therefore, the present inventor recognizes numerous applications of thepresent invention to the $850+million/year process chromatographymarket.

U.S. Pat. No. 4,780,210 to Jen-Chang Hsia entitled “Tangential flowaffinity ultra-filtration” describes a process for trypsin purification.More particularly, it relates to a process of biochemical purificationwhich combines the processing techniques of affinity chromatography andtangential ultra-filtration, and is capable of being operated on acontinuous flow or semi-continuous-flow basis, for use in thepurification (or separation) of molecules of biological interest. Theprocess of the present invention is verifiably different because of thecountercurrent and single-pass nature, along with various otherimprovements. The process described in U.S. Pat. No. 4,780,210 is notsuitable for the biotech market.

Accordingly, while the methods disclosed herein have been described andshown with reference to particular operations performed in a particularorder, it will be understood that these operations may be combined,sub-divided, or re-ordered to form equivalent methods without departingfrom the teachings of the present invention. Accordingly, unlessspecifically indicated herein, the order and grouping of the operationsis not a limitation of the present invention.

Finally, while the invention has been particularly shown and describedwith reference to particular embodiments thereof, it will be understoodby those skilled in the art that various other changes in the form anddetails may be made without departing from the spirit and scope of theinvention, as defined in the appended claims.

What is claimed is:
 1. A system for continuous, single-passcountercurrent tangential negative chromatography having multiplesingle-pass modules, comprising: a single-pass binding step module forbinding impurities from an unpurified product solution with a resinslurry; a single-pass washing step module for washing out a purifiedproduct solution from the resin slurry; a single-pass regeneration stepmodule for regenerating the resin slurry; and a single-passequilibration step module for equilibrating the resin slurry, whereinthe resin slurry flows in a continuous, single-pass through each of thesingle-pass modules, and wherein one or more of the single-pass modulescomprise two or more stages with permeate flow directed countercurrentto resin slurry flow within that single-pass module.
 2. The system ofclaim 1, wherein the system further includes a resin tank for containingthe resin slurry, wherein the resin tank is configured to be reversiblyisolated from the multiple single-pass modules following discharge ofthe resin slurry from the resin tank into the multiple single-passmodules.
 3. The system of claim 1, wherein the permeate flow within eachsingle-pass module is free from pressurization by a permeate pump. 4.The system of claim 1, wherein each single-pass module includes at leastone retentate pump, and the at least one retentate pump is arranged anddisposed to drive and stabilize hydrodynamics of the single-pass module.5. The system of claim 4, wherein each single-pass module includes aplurality of retentate pumps.
 6. The system of claim 1, wherein theresin slurry flows at steady-state through each of the single-passmodules.
 7. The system of claim 1, further including a separate producttank arranged and disposed to capture the purified product solution bycombining and collecting permeate solutions from the single-pass bindingstep module and the single-pass washing step module.
 8. The system ofclaim 1, further including a waste channel arranged and disposed toreceive permeate solutions from the single-pass regeneration step moduleand the single-pass equilibration step module.
 9. The system of claim 1,wherein the system is free of an elution step module.
 10. The system ofclaim 1, wherein the system components are disposable.
 11. A method forcontinuous, single-pass countercurrent tangential negativechromatography having multiple single-pass steps, comprising: receivingunpurified product solution from an upstream process; receiving resinslurry from a resin source; a single-pass binding step for bindingimpurities in the unpurified product solution to the resin slurry fromthe resin source; a single-pass washing step for washing out a purifiedproduct solution from the resin slurry; capturing the purified productsolution from the binding step and the washing step; a single-passregeneration step for regenerating the resin slurry; a single-passequilibration step for equilibrating the resin slurry; and providingbuffer solutions for the single-pass steps, wherein the resin slurryflows in a continuous single pass through each of the single-pass steps,and wherein one or more of the single-pass steps comprise two or morestages with permeate flow directed countercurrent to resin slurry flowwithin that single-pass stage.
 12. The method of claim 11, wherein theresin source includes a resin tank for containing the resin slurry, andthe resin tank is isolated from multiple single-pass modules followingdischarge of the resin slurry from the resin tank into the multiplesingle-pass modules.
 13. The method of claim 11, wherein eachsingle-pass step includes a single-pass module having at least oneretentate pump, and the at least one retentate pump is arranged anddisposed to drive and stabilize hydrodynamics of the single-pass module.14. The method of claim 13, wherein each single-pass module includes aplurality of retentate pumps.
 15. The method of claim 11, wherein thepermeate flow within each single-pass step is free from pressurizationby a permeate pump.
 16. The method of claim 11, wherein the resin slurryflows at steady-state through each of the single-pass steps.
 17. Themethod of claim 11, wherein capturing the purified product solution fromthe binding step and the washing step includes combining and collectingpermeate solutions from the single-pass binding step and the single-passwashing step in a separate product tank.
 18. The method of claim 11,further including directing permeate solutions from the single-passregeneration step and the single-pass equilibration step to waste. 19.The method of claim 11, wherein the method is free of elutionoperations.
 20. The method of claim 11, wherein the purified product isselected from the group consisting of biologics, enzymes, proteins,peptides, small molecules, amino acids, antibiotics, enantiomers, DNA,plasmids, RNA, siRNA, vaccines, polysaccharides, viruses, prions,virus-like particles, plasma proteins, cells, stem cells, andcombinations thereof.